Yutaka Hachimori

States of amino acid residues in proteins: III. Histidine residues in insulin, lysozyme, albumin and proteinases as determined with a new reagent of diazo-i-H-tetrazole

Abstract A new coupling reagent, diazonium- i -H-tetrazole, for spectrophorometric determination of histidine residues in proteins was explored and applied to several proteins. The reagent has the following characteristics advantageous to the determination and is superior to common diazonium compounds. (a) Histidinebisazo- i -H-tetrazole, which is the reaction product to be determined spectrophotometrically, has a strong absorption band at 480 mμ, while tyrosinebisazo- i -H-tetrazole has a weaker band at a considerably longer wavelength, 550 mμ. (b) The coloration due to the formation of biazohistidine residues proceeds to completion before the bis coupling to tyrosine residues. (c) The reagent does not afford colored by-products during its reactions with amino acids or proteins at alkaline pH, so that one can use a high concentration of reagent necessary to determine the end point of coloration. Because of these characteristics, one can determined the molar histidine content simply by absorption photometry at 480 mμ of reaction mixtures of protein and the reagent. The reagent reacts uniformly with all of the histidine residues in bacitracin, lysozyme (EC 3.2.1.17), insulin, albumin and trypsin (EC 3.4.4.4). On the other hand, the reactions with the histidine residues in trypsinogen, chymotrypsinogen and α-chymotrypsin (EC 3.4.4.5) proceed in two steps, in which the reaction in the second step at higher reagent concentrations is due to the coupling to bound histidine residues. The moles of bound residues in the latter proteins were determined and discussed referring to the data obtained previously with other reagents.

States of amino acid residues in proteins: V. Different reactivities with H2O2 of tryptophan residues in lysozyme, proteinases and zymogens

Abstract Hydrogen and organic peroxides were examined in a search for a reagent to differentiate between various states of tryptophan residues in proteins. Hydrogen peroxide in a solution of 0.5 M bicarbonate buffer (pH 8.1–9.4) containing 10% dioxane was found to possess a moderate oxidizing power to oxidize free and bound tryptophan residues at different concentrations of H2O2, and the strength of the tryptophan absorption band of the protein was lowered stepwise with increasing concentration. By the use of this reagent, tryptophan residues in several proteins were classified into various types with different oxidizabilities, and the moles, n, of each type per mole of proteine were determined; n=5 and 1 for lysozyme (EC 3.2.1.17), n=5, 1 and 1 for chymotrypsinogen and α-chymotrypsin (EC 3.4.4.5), and n=2, 1 and 1 for trypsinogen and trypsin (EC 3.4.4.4.) which are arranged in the order of decreasing oxidizability. On the activation of chymotrypsinogen, the oxidizability of the most strongly bound residue decreased greatly whereas, on the activation of trypsinogen, the oxidizability of the secondly oxidized residue increased slightly. The structural rearrangement on the activation was discussed in terms of these changes in state of tryptophan as well as those of other amino acids determined previously. The enzymic activity of lysozyme an proteinases was also measured as a function of H2O2 concentration, and the results were correlated with the degree of oxidation of tryptophan residues. All of the tryptophan residues in denatured proteins were oxidized at a low concentration of H2O2, so that the reagent is applicable for the determination of the molar tryptophan content in protein.

States of amino acid residues in proteins VIII. Tyrosine, histidine and tryptophan residues in chymotrypsin in the presence of substrate and in diisopropylphosphoryl-chymotrypsin

Summary Tyrosine, histidine and tryptophan residues in α-chymotrypsin (EC 3.4.4.5) in the presence and absence of benzoylglycine methyl ester and those in diisopropylphosphoryl-chymotrypsin were analyzed by measuring their reactivities with cyanuric fluoride, diazonium-1-H-tetrazole and H2O2-dioxane, respectively, which are the reagents to differentiate between various states of these residues in protein. By the diisopropylphosphorylation of α-chymotrypsin, two of the total seven tryptophan residues, both of the two histidine residues and one of the four tyrosine residues in the molecule were bound more strongly or burried more deeply. These effects of phosphorylation were discussed from two different view points ; direct effects of the diisopropylphosphoryl group introduced into the specific serine residue and indirect effects of the conformational change induced by the phosphorylation. The addition of benzoylglycine methyl ester to a chymotrypsin solution lowered the reactivity of one of the tyrosine residues with cyanuric fluoride to the same extent as in the phosphorylation, but the histidine and tryptophan residues were unaffected by the presence of substrate. It was inferred from this highly specific effect of substrate that the tyrosine residue is masked by the substrate molecule and is involved in the specific binding of substrate to the enzyme. The activity measured with the same synthetic substrate dropped with pK = 9.6 slightly lower than pK = 10.2 obtained for the ionization of one of the tyrosine residues, and 2 moles of OH− were involved in the inactivation.

Peroxidase activity of hemoproteins. IV. Hematin complexes as model enzymes of peroxidase.

Abstract The process of activation to peroxidase due to the complexing between hematin and chelating reagents (pyridine, histidine, arginine, guanidine, formamide, acetate, and methanol) were studied by measuring both enzymic activity and spectral change as a function of reagent concentration. A large spectral change occurs when a hematin complex carrying two moles of a reagent is formed from hematin. On the other hand, the complexes active as peroxidase carry more than two moles of reagent; two moles on the ferric iron atom of hematin and others, probably, on the protoporphyrin ring. Histidine and arginine are effective in activating hematin, while other essential amino acids are ineffective. The values of k 4 (rate constant for the reaction with hydrogen donor) for the active nonprotein complexes are comparable to or one order less than those for common peroxidases, whereas the values of k 1 (rate constant for the reaction with H 2 O 2 ) are much smaller than the k 1 values for peroxidases. The role of the apoproteins of peroxidases is discussed in connection with the results obtained for the hematin complexes.

The hyperchromic effect of organic denaturing reagents on deoxyribonucleic acid

Abstract 1. 1. The hyperchromic effects of varying concentrations of organic denaturing reagents on the ultraviolet absorption of calf-thymus DNA and of hearing-sperm DNA have been studied at room temperature and over a range of pH values. 2. 2. N , N -Dimethylformamide and N -methylpyrrolidone cause a hyperchromic shift at neutral and alkaline pH values; carboxylic acids (formic, acetic and propionic acids) cause one at acidic pH values; and aliphatic amines (isopropylamine and ethylamine) at alkaline pH values. 3. 3. The percentage increments of the absorbancy at 260 mμ, produced by these reagents, are as high as 49–54% which is approximately equal shift (48–49 %) obtained by heat, and is greater than that (27–31%) obtained by inorganic acid or alkali for the same preparations of DNA. 4. 4. The denaturations effected by these reagents are partially reversible as measured by the hyperchromic shift. 5. 5. The absorbancy-concentrations curves of native DNA show an abrupt change of absorbancy over a narrow range of concentration of reagent which is characteristic of the unique secondary structure of DNA. The curve for the slowly-cooled sample of heat-denatured DNA shows two changes of absorbancy; a linear and gradual increase at low reagent concentrations and an abrupt change at higher concentrations. The curve for the quickly cooled sample shows the linear increase over a wide range of concentration, but no abrupt change.